Blood sampling and testing are routinely carried out for various diagnostic purposes. Detection and quantification of glucose in blood is for example used in the diagnosis and management of disorders of carbohydrate metabolism such as diabetes mellitus. Determination of lactate in blood may for example be used to test acid-base homeostasis and to screen for lactic acidosis, hypoxia or sepsis, and to evaluate adaptation to exercise. An increased level of plasma total homocysteine (tHcy) is for example a risk factor for cardiovascular disease and a sensitive marker for vitamin B deficiencies. In order to be useful diagnostic indicators and predictors, the levels of such blood components should be determined accurately and precisely.
Since metabolism in blood cells (erythrocytes, leukocytes and platelets) is ongoing ex vivo, i.e. after blood collection, levels of blood glucose, lactate and homocysteine can change significantly during the time elapsed between withdrawal and analysis, potentially leading to erroneous results, especially when the time elapsed is variable and uncontrolled and when storage conditions such as temperature vary. Due to continued glycolysis in blood cells, mainly in the erythrocytes, the concentration of blood glucose decreases after collection. Chan et al. in Clinical Chemistry, 38, 1992, pp. 411-413 reported that the plasma glucose concentration in heparinized blood samples at room temperature decreases at a rate of approximately 0.3 mmol/L per hour during the first 12 hours after blood collection. The decrease in blood glucose is accompanied by an increase in lactate concentration. Astles et al. in Clinical Chemistry, 40, 1994, pp. 1327-1330 indicated the increase in lactate to be 0.7 mmol/L per hour, which is large in view of the cited reference interval of 0.5-2.2 mmol/L for lactate. Because of ongoing homocysteine metabolism, wherein homocysteine is formed from methionine via adenosylmethionine-dependent methyl transfer reactions, and ongoing release of homocysteine from erythrocytes, tHcy increases after blood collection. Nauck et al. in Clinical Chemistry and Laboratory Medicine, 39, 2001, pp. 675-680 reported that at room temperature the concentration of tHcy increases by approximately 1-2 μmol/L per hour during the first hours, which corresponds to an approximately 10% increase of tHcy per hour. This means typically after blood collection glucose is continuously consumed by the blood cells and lactate and homocysteine are released from the blood cells.
The changes in glucose, lactate and homocysteine levels in blood after collection are time- and temperature-dependent. Plasma concentration changes of these substances can be prevented by the immediate centrifugation and removal of the blood cells. This requires the addition of anticoagulant, such as EDTA salt, citrate salt, oxalate salt and heparin salt, to the whole blood. Moreover, a centrifuge and the capacity for immediate processing need to be available at or close to the site of blood collection which can be problematic and impractical. Blood samples can be cooled on ice until centrifugation to reduce changes in glucose, lactate and homocysteine levels. However, chilling on ice may not be sufficient and may be impossible or impractical, for example when there is a substantial delay between the collection and the processing and analysis, or when many samples have to be collected and transported. For blood serum, changes in glucose, lactate and homocysteine levels can occur because serum is prepared from a blood sample left at room temperature for a time sufficient to allow coagulation (c. 20-60 min), and centrifugation and separation of the blood cells is possible only thereafter. Preanalytical sample processing can interfere in the subsequent testing for glucose, lactate and homocysteine or co-analytes. For example, disturbance of cellular integrity such as hemolysis may be problematic. Furthermore, time-consuming or error-prone processing steps, for example deproteination using trichloroacetic acid, may not be suitable in routine clinical processing.
When a blood sample cannot be separated or cooled immediately after collection, in addition to an anticoagulant an antiglycolytic agent such as iodoacetate, mannose or fluoride as stabilizer can be added. However, mannose can interfere with enzymatic analysis methods using glucose oxidase or hexokinase. Fluoride stabilizes glucose primarily by inhibiting enolase in the glycolytic pathway. Chan et al. in Clinical Chemistry, 35, 1989, pp. 315-317 showed that fluoride does not prevent loss of plasma glucose completely and that at room temperature the concentration of glucose in blood containing fluoride decreases significantly for the first four hours after collection. Westgard et al. in Clinical Chemistry, 18, 1972, pp. 1334-1338 described that whole blood stabilized with sodium fluoride and separated within 15 min provides an acceptable sample for lactate determination. However, for the case without separation Astles et al. reported that for blood samples with sodium fluoride and potassium oxalate at room temperature lactate increased by 0.2 mmol/L after 1 hour post-collection. Fluoride, adenosine analogues such as 3-deazaadenosine or citrate are used to stabilize tHcy after blood collection, but the use of stabilizers usually leads to deviations at baseline and the problem with stabilization of tHcy is only partly solved. Some typically used stabilizers of tHcy can affect analytical techniques such as fluorescence polarization immunoassay, chemiluminescence immunoassay and enzyme linked immunoassay (see e.g. Nauck et al.).
The use of two agents for the inhibition of glycolysis in blood samples has been considered. Chan et al. (1992) reported the use of sodium fluoride and mannose, but stabilization of the blood glucose concentration was incomplete. Moreover, mannose can interfere with enzymatic analysis methods using glucose oxidase or hexokinase. Bueding and Goldfarb in Journal of Biological Chemistry, 141, 1941, pp. 539-544 demonstrated the use of sodium iodoacetate and sodium fluoride to preserve glucose and lactate. The use of glyceraldehyde and sodium fluoride for the preservation of plasma glucose concentrations was reported by le Roux et al. in Annals of Clinical Biochemistry, 41, 2004, pp. 43-46. Glucose stabilization using sodium fluoride and acidification with citric acid was described by Gambino et al. in Clinical Chemistry, 55, 2009, pp. 1019-1021. However, while these studies aimed at stabilizing glucose and/or lactate, in vitro stabilization of glucose, lactate and homocysteine in blood has not been addressed.
There is a need in the art to make more robust and to simplify preanalytics for the accurate testing of blood glucose, lactate and homocysteine while avoiding cumbersome, complicated, error-prone, costly, impractical or time-critical near-collection processing such as immediate centrifugation or icing of blood samples and providing benefits in terms of improved handling, storage and transport of said samples as well as sample throughput. In particular, the object of the present invention is to effect sufficient and predictable inhibition of glycolysis and to efficiently stabilize blood glucose, lactate and homocysteine in blood after collection at room temperature enabling prolonged storage of blood and allowing blood glucose, lactate and homocysteine and other substances to be determined accurately and reliably using a single sample.